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It is often a primordial feature of neoplasia that, instead of major qualitative changes in the chemical make-up of the cell as a whole, the activity of one or more critical player is 'modified' from being normal. These modifications, which are invariably deregulations, may occur both at the post-transcriptional as well as at the post-translational level and most often conveyance of crucial biochemical signals leading to oncogenic predisposition is through post-translational modifications of the key proteins, especially in the cell nucleus1-5.Affirming the importance of these modifications, there exist plethoras of distinct protein kinases in the genome and the appraisement that majority of all cellular proteins are phosphorylated often at various exclusive sites6, 7. Appropriate regulation of phosphorylation events is essential to the proper function of cellular signaling circuits and even a modest loss of regulation in these pathways results into susceptibility towards many human diseases, including cancer8. Consequently, the enzymes that bring about protein phosphorylation in cell, the kinases, have thus emerged as propitious therapeutic targets in cancer9. Protein kinase CK2 is a profoundly conserved, omnipresently expressed, messenger independent and constitutively (presumably) active serine or threonine kinase that can use ATP as well as GTP as co-substrate localized in cell nucleus and cytoplasm10, 11. In the past decades, myriad evidences have mounted in support of the precedence of protein kinase CK2 in diverse biological processes, particularly stressing its function in controlling cellular growth and proliferation both in normal and diseased contexts13. Particularly, emanating evidences have enunciated about CK2 as a potent suppressor of apoptosis especially in the context of neoplasia, since CK2 has been found to be frequently heightened in most of the cancer systems and almost has become a generalized signature of oncogenesis14-16. One of the significant aspects of CK2 mediated alternation of normal cellular physiology is the nuclear role of CK2, which has been strongly implicated in most cancers. Among its nuclear substrates, are so-called non-histone phospho-protein component of the nucleus is now known to be a mixture of a wide variety of proteins encompassing structural proteins, transcription factors, various enzymes, receptor proteins, and most importantly some crucial tumor-suppressors and regulators of apoptosis17-20. One such critical controller which is targeted for functional disruption by CK2 is the Promyelocytic leukemia (PML), protein which is an essential tumor suppressor21. CK2 regulates PML protein levels by directly phosphorylating it (at residue Ser517) and promoting for ubiquitin-mediated degradation22, 23. PML is the essential component of PML-nuclear bodies (PML-NBs), functioning as the organizing center of this structure and acting as a multi-faceted scaffold for various cellular proteins. PML-NBs are known to modulate various cellular activities, most of them being related to oncogenesis24, 25. Of many such proteins, AKT (PKB) is a potent pro-oncogenic kinase, whose aberrant nuclear functioning is delicately supervised by PML-NBs under normal circumstances, by orchestrating active AKT (phosphorylated form) and its phosphatases, whereby AKT is dephosphorylated, hence inactivated26. This inactivation, coupled with nuclear exclusion of AKT, has a considerable effect on proper functioning of many tumor suppressor proteins, FOXO3a being eminent of them, which is otherwise phosphorylated (at Ser, Thr residues) and deactivated by active nuclear AKT (the ultimate fate of the phosphorylated FOXO3a is proteasomal degradation, following nuclear expulsion)27-30. The ultimate manifestation of the above mentioned signaling dynamics is depressed activity of FOXO3a implicated significantly by uncontrolled cellular proliferation coupled with thwarted apoptosis.
Here, in this study we demonstrate that a finely orchestrated signaling concatenation does operate, commanded by CK2, inflicting of untimely degradation PML, perpetuating abnormal activity of AKT inside the cell nucleus. Inhibition of CK2 activity reverses the above described signalling dynamics manifested by lowered cellular proliferation and apoptosis. Moreover, for the first time, (to the best of our knowledge) we also report that PHLLPP2, another well-established canonical phosphatase of AKT31, 32 interacts with PML in the NBs, effecting dephosphorylation of AKT, thus rendering it inactive which ensures proper functioning of FOXO3a.
Inhibition of CK2 activity significantly affects cellular morphology and migrational ability:
CK2 is well described as a multifunctional protein kinase having vital roles in normal cellular functioning of mammalian cells, including survival and metastasis33, 34. CK2 is aberrantly deregulated in all the malignancies that have been examined till date, including prostate neoplasia35, where CK2 plays a crucial aspect in maintaining oncogenesis. Since in our present study, will be primarily focus on dynamics of CK2 signalling in the context of prostate cancer (PCa), we entertained the idea to ascertain the comprehensive effect of inhibition of CK2 on PC3 prostate cancer cells. CK2 was inhibited in PC3 cells and assayed for the effects on cellular morphology (Figure 1A). We observed that, prostate cancer cells were significantly bereaved of their normal cellular morphology following inhibition of CK2 activity. Cells lost their conventional elongated architecture and assumed a spherical, rounded up appearance, evident of considerable depletion of important survival signalling that was provided by CK2 otherwise. The importance of CK2, is highlighted by the fact that, almost comparable effect was achieved by inhibiting AKT signaling, another well-established cardinal survival factor36. On exploring the effect of CK2 inhibition on cancer cell migration (Figure 1B), we encountered that, even a low but enduring reticence of CK2 signaling impeded PCa cells from usual migrational movement. The fact that needs separate appreciation is that, the detrimental effect of CK2 inhibition is even profound than that of impediment of PI3K/AKT signaling, which reinforces the fact that CK2 is not only an essential homeostatic kinase for cell, but also an indispensible 'onco-kinase' for cancer systems.
Activation of CK2 promotes decline of PML level in prostate cancer cell:
Since in our present study, we are more concerned with pro-oncogenic effect of CK2 in the cell nucleus, tumor suppressor PML became a legitimate target to investigate23. Here we depict that CK2 elicits PML degradation in PC3 prostate cancer cells, as evident from the apparent contrast in the number of PML-NBs, in the conditions where the activity of CK2 is either enhanced (by the d - Sorbitol) treatment or inhibited (by TBCA treatment) (Figure 2A). The experiment was performed for multiple times to quantify the absolute number of PML-NBs with the required statistical significance and the average quantification of PML-NBs, depicting significant dissonance under the differential condition of CK2 activation and inhibition is represented (Figure 2B). Values indubitably depicts that CK2 is acutely responsible for PML degradation, and upon its inhibition, PML is conspicuously stabilized. We also corroborated the microscopy result by inspecting the change in PML protein level by conventional immunoblot analysis under the aforementioned conditions of CK2 activation and inhibition (Figure 2C). Results are perceptibly manifesting of the fact that, PML is stabilized under the situation of CK2 inhibition. In order to evince the time dependent degradation of PML upon over-activation of CK2, steady state level of PML protein was assessed at increasing duration of CK2 activation. It was observed that PML exhibited a gradual loss of protein level over time, most eminent at one hour after the treatment (Figure 2D). A contrasting pattern of gradual stabilization of PML protein level was observed upon increasing duration of CK2 inhibition (Figure 2E S-3a, S-3b). To validate that, indeed CK2 drives PML towards proteasomal degradation, the approach of blockade of the proteasomal system was adapted with simultaneous over expression of CK2 (Figure 2F), since in case PML is degraded via proteasomal system, then, occluding it will prevent the loss of PML protein level, comparable with that of CK2 inhibition. The outcome indeed pointed towards proteasome to be involved degradation of PML.
Tumor suppressor PML interacts with phosphatase PHLPP2 in the nucleus:
Since it has been established that PML-NBs orchestrates a broad spectrum docking scaffold for a multitude of nuclear proteins in order to regulate their activity and AKT/pAKT is prominent of them26, we felt interested to look for whether alike PP2a, PML interacts with another well-established phosphatase, viz. PHLPP2, which is known to dephosphorylate AKT at the Serine 473 residue31 to bring about inactivation of AKT in the nucleus itself. We found PHLPP2 to be interacting with endogenous PML, and the amount of protein thus co-interacted decreases with the decrease in the level of PML, which is obviously due to CK2 mediated degradation (Figure 3A). On the hand, the level of interacting PHLPP2 depicted proportional increment with progressive stabilization of PML, brought about by inhibition of CK2 activity (Figure 3B). Same phenomenon is endorsed by quantification of co-localization co-efficient through time lapse microscopy (Figure 3C), where PHLPP2 depicts an ascertainable increase in co-localization with PML when CK2 is inhibited compared to untreated cells. We have also quantified the change in the level of co-localization, which manifests a pronounced steady increment with increased duration of CK2 inhibition (Figure 3Di). When the co-efficient of co-localization for pAKT-PML was ascertained, it illustrated an antithetical picture, where the co-efficient value for co-localization diminuted uniformly with increasing duration of abatement of CK2 activity (Figure 3Dii). The initial rise in the co-localization level (at 15 mins of TBCA treatment) can be justified by the fact that, PML itself undergo certain degree of stabilization owing to CK2 inhibition, even at that brief exposure to TBCA. But with increasing time and with concomitant stabilization of PML, the level of nuclear pAKT (S473) drops, again supporting our hypothesis that PML-PHLPP2 conglomeration successfully deescalates nuclear AKT, and the decrescence in the level of nuclear pAKT S473 is faithfully reflected by the diminution of the PML-pAKT co-localization co-efficient. Fluorescent Immuno-histochemical analysis of human prostate cancer tissue sample portrays a noticeable level of endogenous co-localization between PHLPP2 and PML (Figure 3E) reinforcing our findings in vivo.
The level of inactive FOXO3a abates with concurrent reduction of active AKT (pAKT) in the nucleus following the CK-PML-PHLPP2 signalling axis:
Pursuing our hypothesis, we next investigated whether the PHLPP2 was indeed effective to dephosphorylate, hence deactivate AKT inside the nucleus. Analysis of pAKT (S473) level in the separate cytoplasmic and nuclear fractions demonstrated that, upon sufficient knock down of both PML and PHLPP2 (Figure 4Ai), active AKT (pAKT S473) gets substantially stabilized in the nuclear pool in comparison to the cytoplasm (Figure 4Aii). Similar observation is achieved after fluorescent microscopy analysis of human prostate cancer cells (PC3), this time activating and inhibiting CK2 (Figure S-4a). It is clearly evident that upon inhibition of CK2 activity, the nuclear pool of active AKT (pAKT S473) wanes noticeably, in contrast to when CK2 activity was enhanced. LY294002 treatment serves as the established control for PI3K/AKT inhibition. Since CK2 exert considerable effect on AKT activation (phosphorylation) by phosphorylation of Ser129 residue of AKT37 (Figure S-4b, upper figure) , we have prepared the CK2 refractory mutant form of AKT viz. AKT-S129D, in which the putative phosphorylation site of CK2 has been rendered constitutive phospho mimetic, hence this mutant form of AKT appears to be insensitive towards change in CK2 activity, as evident from the protein level analysis of pAKT S473 upon both enhancement and inhibition of CK2 activity (Figure S-4b, lower figure). Under normal situation, the localization pattern of AKT-S129D, is chiefly nuclear, owing to its constitutive phospho mimicking, but upon inhibition of CK2 activity, the nuclear quantum of active AKT (pAKT S473) declines appreciably, while augments in the cytoplasmic fraction, as it happens when PML is over expressed (Figure 4C). All the finding collectively enthrusts the existence of an ensembled signalling circuitry commanded by CK2, with PML and PHLPP2 in the interim stairs engaged in the deactivation of nuclear AKT. On assessing the level of phosphorylated FOXO3a (pFOXO3a S253, the inactivated form38), upon knocking down PML and PHLPP2 expression, we discerned that, the amount of pFOXO3a (S253) consequentially elevates (Figure 4D), supposedly due to the loss of the abstainment over active nuclear AKT by PML-PHLPP2. pFOXO3a level is distinctively abated, both upon inhibition of CK2 activity (Figure 4E) and over expression of exogenous PML (Figure 4F), while an utter converse depiction is obtained upon enhancing CK2 activity (Figure 4E), affirmed by exogenous expression of CK2 (Figure 4F), where in both the scenario, the amplitude of pFOXO3a (S253) is significantly heightened. Immuno-fluorescent imaging of PC3 cells elucidates the fact that pFOXO3a level in the nucleus, diminishes conspicuously upon inhibition of CK2 activity (the arrow mark), which is otherwise notably nuclear under CK2 over activation (Figure S-4c). All the aforesaid observation commends that CK2 is considerably responsible for AKT mediated inactivation of FOXO3a, and its subsequent nuclear expulsion through an abrogated PML-PHLPP2 alliance.
Protein Kinase CK2 negatively regulates transcriptional activity of FOXO3a following the (PML -PHLPP2) - AKT - FOXO3a signaling axis:
FOXO3a is one of the cardinal tumor suppressive transcription factor39. The inactivation, coupled with nuclear expulsion of FOXO3a will certainly affect its downstream genes.
p27/Kip1 is among the signature genes, whose expression is governed chiefly by FOXO3a. Analysis of p27/Kip1 and Bim mRNA expression revealed that, both the genes are faithfully transcribed upon PML overexpression and CK2 inhibition, while gets repressed by CK2, and AKT-S129D overexpression (Figure 5A). Congruent results were obtained, when TM-FOXO3a (see figure legend for description) was overexpressed along with inhibition of CK2 activity (Figure 5B) at different duration of time, where mRNA expression is further alleviated due to refrainment of CK2 activity. An interesting result is obtained when gene expression of p27/Kip1 and Bim are quantified upon AKT inhibition alone and followed by CK2 inhibition. The expected rise in the mRNA level of both the genes is further aggrandized due to CK2 inhibition (Figure S5-b). Further, to verify this observation, luciferase reporter assay of p27/Kip1 promoter was carried out. It is clearly evident, both CK2 and AKT-S129D down regulates the promoter transactivation, while the promoter activity was greatly enhanced upon inhibition of CK2 and overexpression of PML (Figure 5C). Noticeably simultaneous inhibition of CK2 along with over-expression of FOXO3a further augments promoter firing, while, CK2 inhibition successfully rolls back the impeding effect of AKT-S129D too. Analogous observation is made with reporter assay with p21/Cip1 (another FOXO3a controlled gene40) gene promoter too (Figure S-5a). The observation at the transcription level was faithfully emulated at the protein level, where both enhancement of CK2 activity and CK2 overexpression detruded the protein expression of p27/Kip1 and Bim, while the protein levels were intensified upon CK2 inhibition and PML overexpression (Figure 5E, 5F). If the increase or decrease in the FOXO3a targets was really due to the PML-PHLPP2-AKT signalling axis, then depreciation in the protein levels of PML andPHLPP2 must affect the transcription and translation of p27/Kip1 and Bim in a negative way. Indeed upon knock down of either PML or PHLPP2, both the mRNA and protein expression of the two genes gets significantly depressed (Figure 5D, 5G). The involvement of nuclear AKT is further reinforced, by the observation that the expression of p27/Kip1 and Bim is hampered upon AKT-S129D overexpression, which is successfully regained by inhibiting CK2 (Figure S-5f). VEGF, a gene whose expression is negatively controlled by FOXO3a41 exhibits a contrapositive pattern of both mRNA and protein expression in comparison to p27/Kip1 and Bim (Figure S5-c, S5-d). All the results collectively indicate that the CK2-PML-(PHLPP-pAKT)-FOXO3a signalling axis is functional even up to the gene expression level, and significantly regulates the expression pattern of crucial FOXO3a targets.
Inhibition of Protein Kinase CK2 significantly arrests cell proliferation followed by apoptosis in cancer cells:
Our previous observations have demonstrated that, inhibition of CK2 stabilizes active FOXO3a in the nucleus, which consequently up-regulates p27/Kip1 and Bim (also p21/Cip1). Among these, p27/Kip1 is well established as a tumor suppressor being a crucial controller of cellular proliferation, also in prostate cancer42, hence its significant augmentation along with Bim, another pivotal factor influencing apoptosis43 is reflected in the analysis of cell cycle. Inhibition of CK2 significantly perturbs cell cycle progression which is evident from the accretion of percentage of cells in the G0/G1 phase. On the other hand, exogenous over expression of CK2, drives the cell back into the state of proliferation (Figure 6A-6B). The higher dose of inhibition even inflicts appreciable amount of cell death, as manifested from the pronounced increase in sub-G0 cell population. The contrasting scenario among cells with empowered CK2 activity and with CK2 activity being inhibited is apparent from the quantification of cell percentage at sub-G0 and G0/G1 phase of cell cycle (Figure 6C). Increased infliction of apoptosis upon inhibition of CK2 activity is agreeably comparable with cells treated with Camptothecin (positive control) as depicted by Annexin-V staining (Figure 6D-6E).
Protein Kinase CK2 is up regulated in in vivo samples of Human Prostate Cancer, while rest of the members in the postulated signalling axis manifests a congruent pattern:
Several reports already exist, advocating the importance of CK2 in PCa, with distinct, mention about oncogenic influence of nuclear CK244. In consistent with that we were interested to reconnaissance the protein level of CK2 in human PCa tissue samples. It is observed that in PCa tissue, not only, the overall protein intensity of CK2 is higher, but also, it is discernibly nuclear (Figure 7A). PML too had been reported to have reduced level of expression in multiple neoplasia45, and we too found that, PML protein expression is perceptibly diminished in PCa samples, in comparison to prostatic hyperplasia (PHp) (Figure 7A). pAKT (S473) and pFOXO3a (S253) both depicted a profuse nuclear staining pattern (in PCa), which is in consistence with our proposed hypothesis (Figure 7A). The staining intensities of CK2 and PML were quantified, which depicts significant difference between PCa and PHp tissues (Figure 7B), moreover when statistically analyzed, it was revealed that, a robust degree of negative correlation (r = - 0.713) exists between them, which again endorses the importance of CK2 mediated degradation of PML. When the staining intensity of all the proteins in the postulated signalling axis (CK2-PML-(PHLPP2-pAKT)-pFOXO3a were estimated to be concurrently analyzed, each of them depicted a significant augmentation of staining intensity in PCa samples when juxtaposed to PHp tissues (Figure 7C). The Mann-Whitney -U test predicts that the difference of H-scores between PCa and PHp samples are indeed statistically significant (Figure 7D, 7E).
The oncogenic influence of protein kinase CK2 has been highlighted consistently in the past decades46. The paramount leverage of CK2 activity in various neoplasias has been profoundly elucidated47. Ample researches have revealed that prostate cancers are predisposed towards up-regulated CK2 and its consequential effects, to such an extent that impeding CK2 signalling is enough to elicit apoptosis48. The aspect which demands special attention in recent times is the pro-oncogenic affairs of nuclear CK2; in the context of prostate cancer44. Tumor suppressor PML has emerged as a pivotal target of intense investigation since it has been demonstrated that CK2 evokes unfledged destruction of PML23, crumbling its imperative role in prohibiting oncogenic transpositions of normal cell21, and control of apoptosis49. Supported by the fact that PML is predominantly modified by posttranslational processes in multitudinous human malignancies22, here we reinforce that CK2 mediated degradation of PML occurs in prostate cancer system too (both cell and tissue). We have successfully identified a second phosphatase isoform (PHLPP2) of AKT to be interacting with PML in PML-NBs, which foster the tumor suppressive activity of PML by supplementing the previously described PML-PP2a coalition26. We have also depicted that PML-PHLPP2 conglomerate convincingly leads to dephosphorylation of AKT inside the nucleus thus rendering it inactive, accounting severe loss of proliferation and survival. PHLPPs already possess a strong reputation for tumor prevention by moderating AKT/PKB31, 50. And our findings will strengthen the pre-existing repute. In relation with prominent role of AKT in prostatic neoplasia51 we illustrated that, deactivating nuclear AKT, accomplishingly abnegate abatement of nuclear FOXO3a, which happens due to AKT mediated phosphorylation on multiple Serine and Threonine aminoacids38. Moreover, since bulk of our present investigation involves androgen independent prostate cancer cells (PC3), the involvement of AKT signalling in the context of androgen impassiveness is re-emphasized52. FOXO3a already has a convincing accolade as tumor suppressor prostate cancer53, 54, our study will invigorate the fact in the scenario of androgen independent PCa. In summary, our data bestow convincing proof, that CK2 plays a decisive role in maintenance and progress androgen independent prostate cancer by promoting precocious destabilization of PML and which effectively abrogate the novel association between PML and PHLPP2. The eventual outcome of which is the perpetuation of AKT activity inside the nucleus causing cessation of FOXO3a activity. Inhibition of CK2, substantially hampers cancer cell migration, proliferation, even induces apoptosis. Although CK2 has been already a target for prostate cancer therapy, much of its demeanor in relation to PML and AKT is still unknown, so do the PML-PHLPP2 coalition dynamics. Analysis of patient sample of PCa establishes our hypothesis evoking a potential possibility of future therapeutic intervention constituting a combination of specific small molecule inhibitor of CK2 and ionizing radiations which is reported to augment cellular FOXO3a level56 in the treatment of androgen refractory prostatic carcinoma with AKT playing pivotal role. Our investigation elucidates an important aspect of cancer cell signalling, by describing the well-orchestrated CK2-PML-PHLPP2-AKT-FOXO3a (Figure 8) network axis that cancer thrives on multiple delicate inter molecular conjunctions, which have evolved complexity to sustain oncogenicity, but at the same time expose the coveted 'Achilles' heel' of cancer.
Conflict of interest
The authors declare no conflict of interests of any kind whatsoever.